Recognition of donor antigens by recipient T cells (allorecognition) initiates the adaptive immune response leading to transplant rejection. It is clear that ideal transplant tolerance protocols should rely on manipulating allorecognition rather than suppressing an ongoing anti-donor immune response. Therefore, elucidation of the mechanisms underlying T cell allorecognition is essential in designing tolerance strategies in transplantation. Our preliminary studies show that, immediately after skin and heart transplantation (day 1), donor-derived vesicles carrying allo-MHC molecules (exosomes) traffic from the graft to the recipient lymphoid organs. Subsequently, recipient cells take up these vesicles and present donor MHC on their surface, a phenomenon called MHC cross-dressing. Our project is to 1) study the cells and molecules involved in this process and 2) investigate the contribution of this phenomenon to T cell allorecognition, alloresponse and allograft rejection. To test this, we propose the following specific aims: Aim 1. To characterize the antigens and cells involved in cross-dressing after transplantation. Aim 2. To test the contribution of exosomes and cross-dressing to alloimmunity and allograft rejection Our project will make use of innovative approaches combining Cre-Lox genetically engineered mouse models, inhibitors of exosome production and flow imaging technologies to investigate the role of donor APCs versus exosome trafficking and MHC cross-dressing in donor antigen presentation, T cell activation and rejection of skin and heart transplants. We anticipate that our proposal will bring new insights into the mechanisms underlying the initiation of alloimmunity and transplant rejection. This knowledge will help design new tolerance protocols in transplantation and potentially autoimmune and other immunological disorders.